614
J. Nat. Prod. 2004, 67, 614-621
Further Labdane and Kaurane Diterpenoids and Other Constituents from Plectranthus fruticosus Cristina Gaspar-Marques,† M. Fa´tima Simo˜es,*,† and Benjamı´n Rodrı´guez*,‡ Faculdade de Farma´ cia da Universidade de Lisboa, Centro de Estudos de Cieˆ ncias Farmaceˆ uticas (CECF), Avenida das Forc¸ as Armadas, 1649-019 Lisboa, Portugal, and Instituto de Quı´mica Orga´ nica, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain Received November 13, 2003
Eight new diterpenoids, one labdane and seven kaurane derivatives, and a new aromadendrane-type sesquiterpenoid have been isolated from the most polar chromatographic fractions of an acetone extract of Plectranthus fruticosus. The structures of the new compounds (1-9) were established mainly by 1D and 2D NMR studies and by some chemical transformations. Compounds 6 and 8 were characterized as their methyl ester derivatives (13 and 14, respectively). Most of the isolated compounds and some of their derivatives were tested as antimicrobial agents, but only 19 showed moderate inhibitory activity against Staphylococcus aureus. Recently,1 we reported the isolation of six new diterpenoids from the less polar chromatographic fractions (eluted with petroleum ether, and 9:1 and 3:1 petroleum etherEtOAc) of an acetone extract of the aerial parts of Plectranthus fruticosus L’He´rit. (Labiatae). In this paper, we report on the isolation and structure elucidation of a new aromadendrane-type sesquiterpenoid (1) and eight additional new diterpenoids, one labdane (2) and seven kaurane derivatives (3-9), all of them found in the more polar chromatographic fractions (eluted with 1:1 petroleum ether-EtOAc and EtOAc) of the acetone extract of the plant. Labdane 101,2 and kaurane 11,3-6 five flavones, and the triterpenoids ursolic and oleanolic acids have also been isolated from the same chromatographic fractions. We also report antimicrobial test results on the isolated compounds and some of their derivatives. Results and Discussion Repeated chromatographic processes on the fractions from the initial chromatography eluted with 1:1 petroleum ether-EtOAc and EtOAc of the acetone extract of P. fruticosus1 (see Experimental Section) yielded compounds 1-11. Compounds 6, 8, and 11 were purified and characterized as their methyl ester derivatives. Apigenin-7,4′dimethyl ether,7 genkwanin,8,9 salvigenin,10,11 cirsimaritin,12 and eupatorin,13,14 and ursolic acid and oleanolic acid15 (characterized as a 2:1 mixture, respectively, of their methyl ester derivatives) were also isolated from the most polar chromatographic fractions of the plant extract. Combustion analysis and low-resolution mass spectrometry indicated a molecular formula C15 H24O2 for 1, and its IR spectrum showed hydroxyl (3429 cm-1) and exocyclic methylene (3076, 1637, 897 cm-1) absorptions. The 1H and 13C NMR spectra of 1 (Experimental Section) were very similar to those reported16,17 for spathulenol [10(14)-aromadendren-4β-ol],18 and the observed differences were consistent with the presence in 1 of a hydroxymethylene group instead of one of the three C-Me groups of spathulenol. The primary alcohol of 1 at the C-15 position18 was in agreement with the diamagnetic shifts observed for its * To whom inquiries should be addressed. (M.F.S.) Tel: +351 217946400. Fax: +352 217946470. E-mail:
[email protected]. (B.R.) Tel: +34 915622900. Fax: +34 915644853. E-mail:
[email protected]. † Faculdade de Farma ´ cia da Universidade de Lisboa, CECF. ‡ Instituto de Quı ´mica Orga´nica, CSIC.
10.1021/np030490j CCC: $27.50
C-3 and C-5 γ-carbons with respect to those of spathulenol16 (∆δ -4.3 and -2.0 ppm, respectively), as well as with the HMBC connectivities between the H2-15 protons and the C-3, C-4, and C-5 carbons of 1. The relative stereochemistry of the H-1R, H-5β, H-6R, and H-7R hydrogens of 1 was supported by the coupling constant values, which were almost identical with those reported19 for 10(14)-aromadendrene. In particular, the observed coupling between the H-1R and H-5β protons (J ) 10.7 Hz) precluded a rings A/B cis junction for 1, because in 10(14)-alloaromadendrene and its derivatives, which
© 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 03/12/2004
Labdane and Kaurane Diterpenoids from Plectranthus
possess a H-1β stereochemistry, this coupling value is 6.67.0 Hz.17,19,20 The aromadendrane-type backbone arrangement of 1 was also in agreement with its 13C NMR spectrum because the C-7, C-8, C-9, and C-14 carbon atom resonances were almost identical with those of spathulenol16 and 10(14)-aromadendrene,19 but very different from those reported for alloaromadendrane-type derivatives, such as in 10(14)-alloaromadendrene.19 An R-configuration for the C-15 hydroxymethylene group of 1 was supported by NOE experiments. Irradiation at δ 0.46 (H-6R proton of 1) caused NOE enhancement in the signals of the H215, H-1R, H-7R, and Me-12 protons, thus establishing that all these hydrogens are on the same side of the plane of the molecule. This result not only substantiated an R-configuration for the hydroxymethylene group of 1 but also confirmed the above established backbone arrangement and allowed the unambiguous assignment of the Me-12 group of this sesquiterpenoid.21,22 Moreover, comparison of the chemical shift of the C-6 and C-12 carbons of 1 [δ 28.46 (CH) and 28.54 (CH3), respectively] with those of spathulenol (δ 30.0 and 26.1, respectively)16 also suggested a 4βhydroxy-4R-hydroxymethylene arrangement, because a diamagnetic shift of the C-6 carbon (∆δ = -1.5 ppm) and a downfield shift of the C-12 carbon (∆δ = +2.4 ppm) with respect to spathulenol have been observed in 4β-hydroxyaromadendrane derivatives possessing an acetoxyl or hydroxyl substituent at the R-side of the molecule, e.g., at the 3R- or 10R-position.22,23 Thus, structure 1 (15-hydroxyspathulenol18) was assigned to the new sesquiterpenoid. The absolute stereochemistry of 1 was not ascertained, although we suppose that it belongs to the normal series like other aromadendranes isolated from higher plants.24 On the contrary, entaromadendranes (and exceptionally normal enantiomers) have been found in red algae, soft corals, marine sponges, and liverworts.24 Several aromadendrane derivatives have been reported among the constituents of the essential oils of Plectranthus species,25 and the hydrocarbon 10(14)aromadendrene occurs in the essential oil of P. fruticosus.26,27 Compound 2 (C22H34O3) showed 1H and 13C NMR spectra almost identical with those of 15, an ent-labdane derivative28 previously found1 in the plant extract. The observed differences between these spectra were consistent with the presence of the acetoxyl group of 2 at the 3β-equatorial position (δH-2β 3.81, 1H, ddd, J ) 11.7, 10.4, 4.3 Hz, δH-3R 4.53, 1H, d, J ) 10.4 Hz) instead of the equatorial 2R-acetate of 15.1,28 The connectivity between the carbonyl carbon of the acetate (δ 172.5) and the proton doublet at δ 4.53 (H-3R), observed in the HMBC spectrum of 2, further supported that 2 and 151 were regioisomers. Alkaline hydrolysis of 2 yielded 10 (C20H32O2), another diterpenoid now isolated in large amounts from P. fruticosus and previously known1 as a synthetic derivative of 15. In fact, 10 had been found for the first time in Croton joufra Roxb. (Euphorbiaceae),2 but its structure had been erroneously established.1,2 An ent-labdane absolute stereochemistry for 10 and 15 has been suggested previously1 on the basis of the change of the molecular rotations. Now, the absolute configuration of all these chemically correlated diterpenoids (2, 10, and 15) was established by using the CD exciton chirality method.29 Benzoylation of 10 yielded 16, the 2R,3β-dibenzoyloxy binary system of which showed a positive first and a negative second Cotton effect (∆235 +12.5, ∆224 -10.3),30 thus defining a positive chirality29 and, consequently, an ent-labdane absolute configuration for 16, and therefore for 2, 10, and 15.
Journal of Natural Products, 2004, Vol. 67, No. 4 615
Another diterpenoid isolated from P. fruticosus (11) was purified as its methyl ester derivative 12, which showed physical and spectroscopic data identical to those reported previously3-6 for methyl ent-12β-hydroxykaur-16-en-19oate.28 Compound 11 has been found in several Compositae species,3,5,6 and it was also obtained4 from grandiflorenic acid. Compound 3 (C22H32O5) and its methyl ester derivative (17, C23H34O5) showed 1H and 13C NMR spectra very similar to those of methyl ent-12β-acetoxykaur-16-en-19oate (18), found as the free acid in the same plant.1 The observed differences in the chemical shifts of the C-7-C-9 and C-13-C-17 carbons of 17 and 18 were in agreement31 with the presence in 17, and hence in 3, of an additional hydroxyl group at the C-15 position. The HMBC spectrum of 3 showed connectivities between the H-15 proton and the C-8, C-9, C-13, C-14, and C-17 carbons, thus confirming the C-15 position for the secondary hydroxyl group. Irradiation at the H-15 proton of 3 and 17 (δ 4.19 and 3.83, respectively) caused NOE enhancements in the signals of the H-9R28 (+11.6 and +9.1%, respectively) protons, thus establishing an R-configuration for the H-15 proton. This NOE experiment not only established the configuration of the C-15 stereogenic center in 3 but also distinguished both methylene protons at C-17 and, more important, precluded the possibility of a phyllocladane (13β-kaurane)32 hydrocarbon skeleton for 3, because the observed NOE between the H-15R and H-9R28 protons is compatible only with a kaurane stereochemistry and not with that of the diastereoisomer phyllocladane, in which the H2-15 and H-9 protons are on opposite sides of the plane of the molecule.32 From all of the above data, it was evident that structure 3 (ent-12β-acetoxy-15β-hydroxykaur-16-en-19-oic acid28) must be assigned to this diterpenoid. The absolute configuration of 3, as well as that of the other new kauranes quoted below (4-9), was not ascertained by direct methods. However, we suppose that 3-9 belong to the enantio series like 11 and other kaurane derivatives found in Plectranthus species.1,25 Moreover, the vast majority of the kaurane-type diterpenoids until now isolated from natural sources belong to the enantio series.32,33 Phyllocladane-type diterpenoids are rare in nature, and they have been isolated predominantly from plants of the Plectranthus (Labiatae)32-35 and Callicarpa (Verbenaceae)36 genera.37 Although several criteria, based on 1H and 13C NMR chemical shifts,32,38 have been used successfully for distinguishing phyllocladanes from kauranes, in this work NOE experiments have shown to be a reliable and easy method for establishing a kaurane hydrocarbon skeleton for 3 (see above) and 4-9 (see below). The 1H and 13C NMR spectra of 4 (C22H32O5) were in agreement with a structure nearly identical with that of 18,1 but possessing a carboxyl function at C-19 instead of the carbomethoxyl group of 18 and an additional secondary alcohol equatorially oriented at the 7β-position.28 The crosspeaks observed in the HMBC spectrum of 4 between the H-7R proton and the C-6, C-8, C-14, and C-15 carbons further confirmed the presence of a 7-hydroxyl substituent in this diterpenoid. Irradiation at δ 3.72 (H-7R proton of 4)28 produced, among others, NOE enhancements in the signals of the H-9R (+15.1%), H-15R (+2.2%), and H-15β (+4.1%) protons. In this compound (4), the NOE observed between the H-7R and H-9R protons does not preclude a phyllocladane-type structure, because these two axial protons are on the same side of the plane of the molecule in both kaurane and phyllocladane stereoisomers. However, the NOEs observed between H-7R and both H2-15
616
Journal of Natural Products, 2004, Vol. 67, No. 4
Table 1.
13C
Gaspar-Marques et al.
NMR Spectral (δ) Data for Compounds 3-5, 8, 9, 13, 17, and 19a
carbon
3b
4b
5c
8d
9b
13d
17d
19d
C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20 -OCOCH3 -OCOCH3 -COOCH3
41.2 (CH2) 19.8 (CH2) 38.6 (CH2) 44.1 (C) 57.0 (CH) 22.0 (CH2) 36.1 (CH2) 47.5 (C) 54.2 (CH) 39.2 (C) 23.5 (CH2) 74.1 (CH) 46.9 (CH) 30.8 (CH2) 83.1 (CH) 157.3 (C) 110.7 (CH2) 29.5 (CH3) 180.2 (C) 14.9 (CH3) 170.1 (C) 21.3 (CH3)
40.9 (CH2) 19.7 (CH2) 38.5 (CH2) 43.83 (C) 53.8 (CH) 32.3 (CH2) 74.55 (CH) 49.8 (C) 55.1 (CH) 39.2 (C) 23.5 (CH2) 74.58 (CH) 48.4 (CH) 25.9 (CH2) 43.83 (CH2) 152.3 (C) 106.4 (CH2) 29.4 (CH3) 180.1 (C) 14.8 (CH3) 170.1 (C) 21.4 (CH3)
41.4 (CH2) 19.9 (CH2) 38.7 (CH2) 43.8 (C) 54.3 (CH) 31.2 (CH2) 75.4 (CH) 56.2 (C) 48.1 (CH) 40.1 (C) 19.5 (CH2) 26.0 (CH2) 44.7 (CH) 35.7 (CH2) 134.6 (CH) 143.0 (C) 15.5 (CH3) 29.2 (CH3) 178.7 (C) 16.1 (CH3)
40.6 (CH2) 18.8 (CH2) 37.9 (CH2) 43.8 (C) 56.4 (CH) 20.5 (CH2) 35.4 (CH2) 42.6 (C) 49.4 (CH) 38.0 (C) 24.1 (CH2) 69.7 (CH) 44.3 (CH) 25.7 (CH2) 67.6 (CH) 59.7 (C) 14.9 (CH3) 28.7 (CH3) 177.8 (C) 13.9 (CH3) 170.3 (C) 21.4 (CH3) 51.2 (CH3)
40.9 (CH2) 19.8 (CH2) 38.5 (CH2) 43.8 (C) 53.7 (CH) 31.4 (CH2) 75.1 (CH) 49.7 (C) 49.0 (CH) 39.6 (C) 18.3 (CH2) 27.7 (CH2) 39.0 (CH) 24.9 (CH2) 67.0 (CH) 59.5 (C) 14.8 (CH3) 29.3 (CH3) 180.0 (C) 15.9 (CH3)
40.2 (CH2) 18.8 (CH2) 37.8 (CH2) 43.6 (C) 53.2 (CH) 29.5 (CH2) 75.2 (CH) 54.2 (C) 47.3 (CH) 37.9 (C) 24.9 (CH2) 69.2 (CH) 48.5 (CH) 28.8 (CH2) 135.1 (CH) 144.2 (C) 15.8 (CH3) 28.6 (CH3) 177.7 (C) 13.3 (CH3) 170.6 (C) 21.6 (CH3) 51.2 (CH3)
40.6 (CH2) 18.9 (CH2) 37.8 (CH2) 43.8 (C) 56.7 (CH) 20.8 (CH2) 34.9 (CH2) 46.7 (C) 53.4 (CH) 38.4 (C) 23.0 (CH2) 73.3 (CH) 46.1 (CH) 30.1 (CH2) 83.0 (CH) 155.5 (C) 111.7 (CH2) 28.8 (CH3) 178.0 (C) 13.9 (CH3) 170.4 (C) 21.5 (CH3) 51.2 (CH3)
40.4 (CH2) 18.8 (CH2) 37.78 (CH2) 43.8 (C) 56.2 (CH) 20.1 (CH2) 37.78 (CH2) 49.7 (C) 46.2 (CH) 38.4 (C) 24.6 (CH2) 68.0 (CH) 42.3 (CH) 36.6 (CH2) 163.4 (CH) 147.1 (C) 188.4 (CH) 28.7 (CH3) 177.7 (C) 13.3 (CH3) 170.1 (C) 21.4 (CH3) 51.3 (CH3)
a At 100 MHz. All these assignments were in agreement with HSQC and HMBC spectra. b In pyridine-d solution. c In acetone-d 5 6 solution. d In CDCl3 solution.
protons clearly established that 4 possessed a kauranetype structure, in which these hydrogens are on the same side of the molecule, whereas they are on opposite sides in the phyllocladane hydrocarbon skeleton.32 In addition, irradiation at δ 3.30 (H-15β proton of 4) caused a strong NOE enhancement (+4.4%) in the signal of HA-17. Consequently, compound 4 was formulated as ent-12β-acetoxy7β-hydroxykaur-16-en-19-oic acid.28 The 1H and 13C NMR spectra of compound 5 (C20H30O3) showed signals for a C-19 carboxyl group and a 7β-hydroxyl substituent oriented equatorially as in 4. This conclusion was supported by the HMBC spectrum of 5, which displayed connectivities compatible only with the proposed structure (e.g., between the H-7R proton and the C-14 and C-15 carbons, and between the C-7 carbon and the H-5R, H2-6, and H2-14 protons, as well as between the carboxyl carbon at C-19 and the H2-3, H-5R, and Me-18 protons). Irradiation at the olefinic proton of 5 (δ 5.14, H-15) caused NOE enhancements in the signals of the H-9R (+2.0%) and H-7R (+5.0%) protons, thus confirming a kaurane-type structure for 5 and located its secondary hydroxyl group at the 7β-position. Therefore, compound 5 is ent-7β-hydroxykaur-15-en-19-oic acid.28 The kaur-15-ene derivative 6 was also found in the acetone extract of P. fruticosus, and it was purified as its methyl ester derivative 13 (C23H34O5). The 1H and 13C NMR spectra of this substance (13: methyl ent-12β-acetoxy-7βhydroxykaur-15-en-19-oate)28 were similar to those of 4, showing characteristic signals for a kaur-15-ene derivative instead of the kaur-16-ene structure of 4. Moreover, the observed differences in the chemical shifts of the C-6-C9, C-12, and C-14 carbons of 13 and 4 (Table 1) further supported31 the structure of the former. The kaurane-type structure of 13 was also in agreement with the NOE observed for the H-9R signal28 (+2.1% NOE enhancement) when the signal of the H-15 proton (δ 5.19) was irradiated. Compound 7 was transformed into its methyl ester derivative 19 (C23H32O5) by treatment with an ethereal solution of diazomethane. The IR and UV spectra of 19 showed absorptions typical for an R,β-unsaturated aldehyde function, and its kaur-15-en-17-al partial structure was supported39-42 by the 1H and 13C NMR data. In
addition, the 1H NMR spectra of 7 and 19, as well as the 13C NMR spectrum of 19 (Table 1), revealed the presence of a 12β-acetoxyl substituent in both compounds, identical with that found in 13, and a carbomethoxyl group at the C-19 position, which is a carboxylic acid in 7. The HMBC spectrum and other spectroscopic data of 19 were in agreement with a structure of ent-12β-acetoxy-17-oxokaur15-en-19-oic acid28 for this new diterpenoid (7). Compound 8 was characterized as its methyl ester derivative 14 (C23H34O5). The 1H and 13C NMR spectra of 14 were very similar to those reported1 for 20. The observed differences between the 1H and 13C NMR spectra of 14 and 20 were consistent with the presence in the former of a 12β-acetoxyl substituent instead of the C-12 methylene of the later. This was strongly supported by the HMBC spectrum of 14 (connectivities between the C-12 carbon and the H-9R, H2-11, H-13β, and H-14R protons) and by the observed downfield shifts of the C-11-C-13 (∆δ +5.9, +42.7, and +5.3 ppm, respectively) and upfield shifts of the C-14 and C-16 (∆δ -6.3 and -1.7) carbons of 14 (Table 1) with respect to those of 20.1 In addition, irradiation at δ 4.97 (H-12R of 14) produced strong NOE enhancements in the signals of the H-11R (+4.8%) and Me-17 (+4.7%) protons, whereas on irradiating at δ 2.63 (H-15R of 14) the signals of the H-7R, H-9R, H-11R, and Me-17 protons were enhanced (+1.8, +4.1, +1.1, and +3.3%, respectively). These NOE results established 15β,16β-stereochemistry for the oxirane and confirmed a kaurane-type structure for 14, which must be methyl ent-12β-acetoxy-15β,16β-epoxykauran-19-oate.28 Compound 9 (C20H30O4) possessed a 15,16-epoxyde [δH 2.91 (1H, s, H-15R) and 1.41 (3H, s, Me-17); δC 67.0 (CH, C-15), 59.5 (C, C-16), and 14.8 (CH3, C-17)] as in 14 (see above) and 20.1 The 1H and 13C NMR spectra of 9 were in agreement with the presence of a C-19 carboxyl group and a secondary hydroxyl group at the C-7β equatorial position such as in 4, 5, and 13. The ent-7β-hydroxy-15β,16βepoxykauran-19-oic acid28 structure for 9 was also supported by the observed HMBC cross-peaks between the H-7R proton and the C-5, C-6, C-8, C-9, C-14, and C-15 carbons and by the NOE caused on the H-7R, H-9R, and Me-17 proton signals (+4.1, +3.9, and +2.3% NOE en-
Labdane and Kaurane Diterpenoids from Plectranthus
hancement, respectively) when the H-15R proton of 9 (δ 2.91) was irradiated. Compounds 1, 3-5, 7, 9, 10, 12-14, 17, and 19 were tested for antimicrobial activity against Gram-positive and Gram-negative bacteria and yeast strains (see Experimental Section). None of the compounds showed activity against Pseudomonas aeruginosa, Escherischia coli, and Candida albicans strains. Against Staphylococcus aureus only the kaurane 19 showed moderate activity (MIC value 62.5 µg/mL). Experimental Section General Experimental Procedures. Melting points were determined on a Kofler block and are uncorrected. Optical rotations were measured on a Perkin-Elmer 241 MC polarimeter. UV spectra were recorded on a Perkin-Elmer Lambda 2 UV/vis spectrophotometer. IR spectra were obtained on a Perkin-Elmer Spectrum One spectrophotometer. 1H and 13C NMR spectra were recorded in CDCl3 (1, 2, 7, 10, 12-14, 16, 17, and 19), pyridine-d5 (3, 4, and 9), or acetone-d6 (5) solution on a Varian INOVA 400 apparatus at 400 and 100 MHz, respectively, except for 7 (1H NMR at 300 MHz, Varian INOVA 300 apparatus). Chemical shifts are reported with respect to residual CHCl3 (δ 7.25) or pyridine-d5 (δ 8.71, 7.55, 7.19) or acetone-d6 (δ 2.04) signals for protons and to the solvent signals (δCDCl3 77.00, δpyridine-d5 149.9, 135.5, 123.5, δacetone-d6 206.1, 29.8) for carbons. All the assignments for protons and carbons were in agreement with 2D COSY, TOCSY, gHSQC, gHMBC, and 1D NOESY spectra. Mass spectra were registered in the positive EI mode on a Hewlett-Packard 5973 instrument (70 eV). Elemental analyses were conducted on a Carlo Erba EA 1108 apparatus. Merck Si gel (70-230 mesh and 230-400 mesh, for gravity flow and flash chromatograpy, respectively) was used for column chromatography. Merck 5554 Kieselgel 60 F254 sheets were used for TLC analysis. Petroleum ether (bp 50-70 °C) was used for column chromatography. Plant Material. Plectranthus fruticosus was cultivated in the Faculty of Pharmacy Hortum, Lisbon University, from seeds provided by the Herbarium of the Botanical Garden of Lisbon, Portugal. Aerial parts of this species were collected in June 1999, and voucher specimens were deposited in the Herbarium of the Botanical Center of the “Instituto de Investigac¸ a˜o Cientı´fica Tropical”, Lisbon (ref C. Marques, S/N° LISC). Extraction and Isolation. Dried and powdered P. fruticosus aerial parts (3.58 kg) were extracted with Me2CO as described previously.1 A part (100 g) of the total extract (444 g) was subjected to column chromatography (Si gel 70-230 mesh, 960 g) eluting successively with petroleum ether, petroleum ether-EtOAc (9:1, 3:1, and 1:1), and EtOAc. The constituents of the chromatographic fractions eluted with 9:1 and 3:1 petroleum ether-EtOAc have previously been reported,1 and those eluted with 1:1 petroleum ether-EtOAc and EtOAc were isolated as follows. The residue (20.3 g) of the fractions eluted with 1:1 petroleum ether-EtOAc was rechromatographed (Si gel 230400 mesh column, 800 g, eluted with a petroleum ether-EtOAc gradient from 8.5:1.5 to 3:7). The fraction eluted with 8.5:1.5 petroleum ether-EtOAc yielded impure 2 (30 mg), which was rechromatographed (Si gel 230-400 mesh, 20 g, 7:1 petroleum ether-EtOAc as eluent), affording pure 2 (12 mg, 0.0015% on dry plant material). The fraction eluted with 8.2:1.8 petroleum ether-EtOAc yielded apigenin-7,4′-dimethyl ether7 (5-hydroxy7,4′-dimethoxyflavone, 3 mg, 0.00037%). The residue (4 g) of the fractions eluted with 4:1 petroleum ether-EtOAc was treated with an excess of an ethereal solution of CH2N2 at room temperature for 3 h and then subjected to column chromatography (Si gel 230-400 mesh, 200 g, 9:1 CH2Cl2-EtOAc as eluent), giving the following compounds in order of increasing polarity: 14 (methyl ester of 8, 197 mg, 0.024%), a 2:1 mixture of the methyl esters of ursolic and oleanolic acids15 (255 mg,
Journal of Natural Products, 2004, Vol. 67, No. 4 617
0.032%), the aromadendrene derivative 1 (4 mg, 0.0005%), and 101,2 (590 mg, 0.073%). The residue (1.1 g) of the fractions eluted with 3:1 petroleum ether-EtOAc was rechromatographed (Si gel 230-400 mesh column, 120 g, eluted with 8.2: 1.5 CH2Cl2-EtOAc), yielding, in order of increasing polarity, genkwanin8,9 (5,4′-dihydroxy-7-methoxyflavone, 3 mg, 0.00037%), 7 (28 mg, 0.0034%), and a mixture of 7 and 11. This mixture was methylated with an ethereal solution of CH2N2 and then subjected to column chromatography (Si gel 230-400 mesh, 40 g, eluted with 3:1 petroleum ether-EtOAc), affording the methyl ester of 7 (19, 26 mg, 0.0032%) and the previously known3-6 kaurane derivative 12 (7 mg, 0.0009%). The fractions eluted with 7:3 petroleum ether-EtOAc contained 630 mg of a complex mixture of compounds. This mixture was rechromatographed (Si gel 230-400 mesh column, 140 g, eluted with 3:1 CH2Cl2-EtOAc), yielding the following compounds in order of increasing polarity: salvigenin10,11 (5-hydroxy-6,7,4′-trimethoxyflavone, 188 mg, 0.023%), 5 (3 mg, 0.00037%), 3 (68 mg, 0.0084%), cirsimaritin12 (5,4′dihydroxy-6,7-dimethoxyflavone, 7 mg, 0.0009%), and 4 (37 mg, 0.0046%). Finally, the fractions eluted with 3:7 petroleum ether-EtOAc (720 mg) yielded, after rechromatography (Si gel 230-400 mesh column, 80 g, 97:3 CH2Cl2-MeOH as eluent), 30 mg of an impure compound (6), which was treated with an excess of an ethereal solution of CH2N2 for 3 h and then chromatographed (Si gel 230-400 mesh column, 20 g, 3:1 petroleum ether-EtOAc as eluent), giving pure 13 (25 mg, 0.0031%). The fractions from the initial chromatography eluted with EtOAc gave a residue (21.5 g). Rechromatography of this residue (Si gel 230-400 mesh column, 350 g, 1:1 CH2Cl2EtOAc as eluent) successively afforded eupatorin13,14 (5,3′dihydroxy-6,7,4′-trimethoxyflavone, 19 mg, 0.0024%) and 9 (100 mg, 0.012%). The previously known flavones (apigenin-7,4′-dimethyl ether,7 genkwanin,8,9 salvigenin,10,11 cirsimaritin,12 and eupatorin13,14) were identified by their mp and 1H NMR spectra, and the mixture of ursolic and oleanolic acid methyl esters was characterized by a careful study15 of the 1H NMR spectrum and by comparison (TLC) with authentic samples. 10(14)-Aromadendrene-4β,15-diol (1): colorless needles (spontaneously on cooling), mp 73-75 °C; [R]D18 +9.3° (c 0.215, CHCl3); IR (KBr) νmax 3429, 3076, 2922, 2862, 1637, 1456, 1375, 1090, 1026, 897 cm-1; 1H NMR (CDCl3, 400 MHz) δ 4.70 (1H, t, J14B,14A ) J14B,1R ) 1.8 Hz, HB-14), 4.67 (1H, dd, J14A,14B ) 1.8 Hz, J14A,9R ) 0.8 Hz, HA-14), 3.70 (1H, d, J15B,15A ) 11.2 Hz, HB-15), 3.57 (1H, d, J15A,15B ) 11.2 Hz, HA-15), 2.41 (1H, ddd, J9β,9R ) 13.6 Hz, J9β,8R ) 6.4 Hz, J9β,8β ) 1.2 Hz, H-9β), 2.18 (1H, dddd, J1R,2R ) 6.0 Hz, J1R,2β ) 12.0 Hz, J1R,5β ) 10.7 Hz, J1R,14B ) 1.8 Hz, H-1R), 2.00 (1H, dddd, J9R,9β ) 13.6 Hz, J9R,8R ) 1.1 Hz, J9R,8β ) 12.3 Hz, J9R,14A ) 0.8 Hz, H-9R), 1.96 (1H, dddd, J8R,8β ) 13.6 Hz, J8R,7R ) 6.0 Hz, J8R,9R ) 1.1 Hz, J8R,9β ) 6.4 Hz, H-8R), 1.91 (1H, dddd, J2β,2R ) 13.1 Hz, J2β,1R ) 12.0 Hz, J2β,3R ) 13.0 Hz, J2β,3β ) 6.0 Hz, H-2β), 1.90 (1H, br s, 15-OH), 1.81 (1H, ddd, J3β,3R ) 13.0 Hz, J3β,2R ) 1.2 Hz, J3β,2β ) 6.0 Hz, H-3β), 1.69 (1H, dddd, J2R,2β ) 13.1 Hz, J2R,1R ) 6.0 Hz, J2R,3R ) 6.0 Hz, J2R,3β ) 1.2 Hz, H-2R), 1.58 (1H, br s, 4βOH), 1.51 (1H, td, J3R,3β ) J3R,2β ) 13.0 Hz, J3R,2R ) 6.0 Hz, H-3R), 1.36 (1H, dd, J5β,1R ) 10.7 Hz, J5β,6R ) 11.2 Hz, H-5β), 1.05 (3H, s, Me-12), 1.04 (3H, s, Me-13), 0.98 (1H, dddd, J8β,8R ) 13.6 Hz, J8β,7R ) 11.3 Hz, J8β,9R ) 12.3 Hz, J8β,9β ) 1.2 Hz, H-8β), 0.73 (1H, ddd, J7R,6R ) 9.5 Hz, J7R,8R ) 6.0 Hz, J7R,8β ) 11.3 Hz, H-7R), 0.46 (1H, dd, J6R,5β ) 11.2 Hz, J6R,7R ) 9.5 Hz, H-6R); 13C NMR (CDCl3, 100 MHz) δ 152.7 (C, C-10), 107.0 (CH2, C-14), 82.9 (C, C-4), 68.3 (CH2, C-15), 53.9 (CH, C-1), 52.4 (CH, C-5), 38.6 (CH2, C-9), 37.5 (CH2, C-3), 28.54 (CH3, C-12), 28.46 (CH, C-6), 27.6 (CH, C-7), 27.2 (CH2, C-2), 24.4 (CH2, C-8), 20.5 (C, C-11), 16.1 (CH3, C-13); EIMS m/z 236 [M]+ (1), 218 (29), 205 (100), 203 (52), 187 (61), 175 (25), 162 (30), 149 (44), 147 (44), 145 (39), 133 (44), 131 (52), 119 (55), 107 (55), 105 (61), 93 (58); anal. C 76.34%, H 10.28%, calcd for C15H24O2, C 76.23%, H 10.23%. ent-3β-Acetoxylabda-8(17),12Z,14-trien-2r-ol (2):28 amorphous white solid, mp 105-115 °C; [R]D18 -16.5° (c 0.291, CHCl3); UV (MeOH) λmax (log ) 234 (3.82) nm; IR (KBr) νmax
618
Journal of Natural Products, 2004, Vol. 67, No. 4
3437, 3082, 2939, 2851, 1736, 1643, 1439, 1371, 1246, 1057, 1030, 958, 890 cm-1; 1H NMR (CDCl3, 400 MHz) δ 6.76 (1H, ddd, J14,15A ) 10.8 Hz, J14,15B ) 17.2 Hz, J14,12 ) 0.8 Hz, H-14), 5.28 (1H, br t, J12,11A ) J12,11B ) 6.5 Hz, H-12), 5.18 (1H, ddd, J15B,15A ) 1.6 Hz, J15B,14 ) 17.2 Hz, J15B,12 ) 0.8 Hz, pro-Z HB15), 5.09 (1H, dt, J15A,15B ) J15A,12 ) 1.6 Hz, J15A,14 ) 10.8 Hz, pro-E HA-15), 4.87 (1H, q, J17B,17A ) J17B,7R ) J17B,9R ) 1.6 Hz, pro-E HB-17), 4.53 (1H, d, J3R,2β ) 10.4 Hz, H-3R), 4.52 (1H, q, J17A,17B ) J17A,7R ) J17A,9R ) 1.6 Hz, pro-Z HA-17), 3.81 (1H, ddd, J2β,1R ) 11.7 Hz, J2β,1β ) 4.3 Hz, J2β,3R ) 10.4 Hz, H-2β), 2.42 (1H, m*, HB-11), 2.40 (1H, ddd, J7β,7R ) 13.2 Hz, J7β,6R ) 2.4 Hz, J7β,6β ) 4.2 Hz, H-7β), 2.21 (1H, dd, J1β,1R ) 12.6 Hz, J1β,2β ) 4.3 Hz, H-1β), 2.19 (1H, ddd, J11A,11B ) 17.5 Hz, J11A,9R ) 11.0 Hz, J11A,12 ) 6.5 Hz, HA-11), 2.14 (3H, s, 3β-OAc), 2.00 (1H, br ddd, J7R,7β ) 13.2 Hz, J7R,6R ) 5.2 Hz, J7R,6β ) 12.7 Hz, H-7R), 1.76 (3H, d, J16,12 ) 1.2 Hz, Me-16), 1.74 (1H, br dd, J9R,11A ) 11.0 Hz, J9R,11B ) 3.3 Hz, H-9R), 1.70 (1H, m*, H-6R), 1.58 (1H, br, 2R-OH), 1.38 (1H, m*, H-6β), 1.26 (2H, m*, H-1R and H-5R), 0.89 (3H, s, Me-18), 0.86 (3H, s, Me-19), 0.79 (3H, s, Me-20);43 13C NMR (CDCl3, 100 MHz) δ 172.5 (C, OCOCH3), 146.9 (C, C-8), 133.7 (CH, C-14), 132.0 (C, C-13), 130.7 (CH, C-12), 113.6 (CH2, C-15), 108.9 (CH2, C-17), 84.5 (CH, C-3), 67.9 (CH, C-2), 56.9 (CH, C-9), 54.4 (CH, C-5), 46.3 (CH2, C-1), 40.1 (C, C-10), 39.3 (C, C-4), 37.6 (CH2, C-7), 28.7 (CH3, C-18), 23.5 (CH2, C-6), 22.3 (CH2, C-11), 21.2 (CH3, OCOCH3), 19.7 (CH3, C-16), 17.5 (CH3, C-19), 15.4 (CH3, C-20); EIMS m/z 346 [M]+ (0.5), 331 (1), 286 (1), 271 (3), 253 (2), 187 (8), 149 (14), 137 (14), 135 (16), 133 (23), 109 (27), 107 (23), 43 (100); anal. C 76.41%, H 9.69%, calcd for C22H34O3, C 76.26%, H 9.89%. ent-12β-Acetoxy-15β-hydroxykaur-16-en-19-oic acid (3):28 colorless hexagonal plates (EtOAc-n-pentane), mp 244-246 °C; [R]D20 -58.5° (c 0.301, MeOH); IR (KBr) νmax 3415, 3065, 2950, 2850, 1740, 1703, 1636, 1449, 1371, 1228, 1014, 999, 964, 906 cm-1; 1H NMR (pyridine-d5, 400 MHz) δ 14.70 (1H, br, 19-COOH), 5.90 (1H, br, 15β-OH), 5.52 (1H, dd, J17B,17A ) 1.2 Hz, J17B,15R ) 0.8 Hz, HB-17, cis hydrogen with respect to C-15), 5.28 (1H, dd, J17A,17B ) 1.2 Hz, J17A,15R ) 1.6 Hz, HA-17, trans hydrogen with respect to C-15), 4.97 (1H, br t, J12R,11R ) 4.5 Hz, J12R,11β < 0.5 Hz, J12R,13β ) 4.6 Hz, H-12R), 4.19 (1H, br s, H-15R), 2.97 (1H, ddd, J13β,12R ) 4.6 Hz, J13β,14R ) 5.0 Hz, J13β14β ) 0.5 Hz, H-13β), 2.50 (1H, dddd, J3β,3R ) 13.2 Hz, J3β,2R ) 4.1 Hz, J3β,2β ) 3.6 Hz, J3β,1β ) 0.8 Hz, H-3β), 2.32 (1H, br dd, J14β,14R ) 11.4 Hz, J14β,13β ) 0.5 Hz, J14β,15R ) 1.0 Hz, H-14β), 2.26 (4H, m*, H-2β, H-6R, H-6β, and H-7β), 1.98 (3H, s, 12βOAc), 1.85 (2H, m*, H-7R and H-11R), 1.81 (1H, m*, H-1β), 1.78 (1H, br d, J11β,11R ) 16.8 Hz, J11β,9R = J11β,12R < 0.5 Hz, H-11β), 1.57 (1H, dd, J14R,14β ) 11.4 Hz, J14R,13β ) 5.0 Hz, H-14R), 1.49 (1H, ddddd, J2R,2β ) 14.0 Hz, J2R,1R ) 4.1 Hz, J2R,1β ) 3.2 Hz, J2R,3R ) 4.4 Hz, J2R,3β ) 4.1 Hz, H-2R), 1.40 (3H, s, Me-20), 1.38 (3H, s, Me-18), 1.31 (1H, br d, J9R,11R ) 8.8 Hz, J9R,11β < 0.5 Hz, H-9R), 1.18 (1H, dd, J5R,6R ) 3.8 Hz, J5R,6β ) 10.2 Hz, H-5R), 1.08 (1H, td, J3R,3β ) J3R,2β ) 13.2 Hz, J3R,2R ) 4.4 Hz, H-3R), 0.82 (1H, ddd, J1R,1β ) 13.2 Hz, J1R,2R ) 4.1 Hz, J1R,2β ) 12.8 Hz, H-1R);43 13C NMR (pyridine-d5, 100 MHz), see Table 1; EIMS m/z 376 [M]+ (1), 334 (1), 316 (100), 301 (38), 298 (20), 283 (17), 270 (30), 255 (19), 243 (12), 237 (15), 199 (11), 197 (13), 183 (10), 173 (14), 161 (25), 160 (20), 148 (30), 145 (25), 133 (24), 131 (29), 123 (27), 121 (34), 109 (34), 105 (40); anal. C 70.39%, H 8.61%, calcd for C22H32O5, C 70.18%, H 8.57%. ent-12β-Acetoxy-7β-hydroxykaur-16-en-19-oic acid (4):28 colorless fine needles (EtOAc-n-pentane), mp 250-252 °C; [R]D20 -52.1° (c 0.313, MeOH); IR (KBr) νmax 3419, 3071, 2940, 2873, 1736, 1691, 1635, 1467, 1372, 1238, 1028, 968, 876 cm-1; 1H NMR (pyridine-d , 400 MHz) δ 6.15 (1H, br, 7β-OH), 5.05 5 (1H, ddd, J17B,17A ) 1.2 Hz, J17B,15R ) 0.5 Hz, J17B,15β ) 2.3 Hz, HB-17, trans hydrogen with respect to C-15), 5.01 (1H br dd, J12R,11R ) 5.8 Hz, J12R,11β < 0.5 Hz, J12R,13β ) 5.0 Hz, H-12R), 4.95 (1H, ddd, J17A,17B ) 1.2 Hz, J17A,15R ) 0.5 Hz, J17A,15β ) 2.3 Hz, HA-17, cis hydrogen with respect to C-15), 3.72 (1H, dd, J7R,6R ) 4.5 Hz, J7R,6β ) 11.3 Hz, H-7R), 3.30 (1H, dt, J15β,15R ) 17.0 Hz, J15β,17A ) J15β,17B ) 2.3 Hz, H-15β), 2.94 (1H, m, W1/2 ) 9 Hz, H-13β), 2.62 (1H, ddd, J6R,6β ) 13.6 Hz, J6R,5R ) 2.4 Hz, J6R,7R ) 4.5 Hz, H-6R), 2.57 (1H, ddd, J6β,6R ) 13.6 Hz, J6β,5R ) 12.0 Hz, J6β,7R ) 11.3 Hz, H-6β), 2.50 (1H, dddd, J3β,3R )
Gaspar-Marques et al.
13.5 Hz, J3β,2R ) 2.8 Hz, J3β,2β ) 3.5 Hz, J3β,1β ) 1.0 Hz, H-3β), 2.26 (1H, dddt, J2β,2R ) 13.6 Hz, J2β,1R ) 13.1 Hz, J2β,3R ) 13.5 Hz, J2β,1β ) J2β,3β ) 3.5 Hz, H-2β), 2.14 (2H, br s, H-14R and H-14β), 2.12 (1H, br d, J15R,15β ) 17.0 Hz, J15R,17A ) J15R,17B ) 0.5 Hz, H-15R), 2.01 (1H, ddd, J11R,11β ) 16.9 Hz, J11R,9R ) 9.2 Hz, J11R,12R ) 5.8 Hz, H-11R), 1.95 (3H, s, 12β-OAc), 1.83 (1H, br d, J11β,11R ) 16.9 Hz, J11β,9R ) J11β,12R < 0.5 Hz, H-11β), 1.76 (1H, dddd, J1β,1R ) 13.1 Hz, J1β,2R ) 3.0 Hz, J1β,2β ) 3.5 Hz, J1β,3β ) 1.0 Hz, H-1β), 1.50 (1H, ddddd, J2R,2β ) 13.6 Hz, J2R,1R ) 3.6 Hz, J2R,1β ) 3.0 Hz, J2R,3R ) 4.2 Hz, J2R,3β ) 2.8 Hz, H-2R), 1.44 (3H, s, Me-20), 1.38 (3H, s, Me-18), 1.30 (1H, br d, J9R,11R ) 9.2 Hz, J9R,11β < 0.5 Hz, H-9R), 1.27 (1H, dd, J5R,6R ) 2.4 Hz, J5R,6β ) 12.0 Hz, H-5R), 1.09 (1H, td, J3R,3β ) J3R,2β ) 13.5 Hz, J3R,2R ) 4.2 Hz, H-3R), 0.82 (1H, td, J1R,1β ) J1R,2β ) 13.1 Hz, J1R,2R ) 3.6 Hz, H-1R); 13C NMR (pyridine-d5, 100 MHz), see Table 1; EIMS m/z 376 [M]+ (1), 358 (11), 343 (1), 340 (1), 334 (1), 316 (43), 301 (12), 298 (100), 283 (15), 273 (84), 253 (18), 237 (15), 227 (15), 197 (17), 183 (19), 171 (14), 162 (88), 145 (35), 144 (37), 133 (23), 131 (27), 123 (42), 119 (34), 117 (24), 109 (31), 107 (42), 105 (42); anal. C 70.02%, H 8.71%, calcd for C22H32O5, C 70.18%, H 8.57%. ent-7β-Hydroxykaur-15-en-19-oic acid (5):28 colorless fine needles (EtOAc-n-pentane), mp 266-268 °C; [R]D20 -58.2° (c 0.146, MeOH); IR (KBr) νmax 3417, 2932, 2872, 1697, 1469, 1295, 1251, 1237, 1193, 1057, 1000, 898, 811 cm-1; 1H NMR (acetone-d6, 400 MHz) δ 5.14 (1H, q, J15,17 ) 1.5 Hz, H-15), 3.48 (1H, dd, J7R,6R ) 3.9 Hz, J7R,6β ) 11.7 Hz, H-7R), 2.31 (1H, m, W1/2 ) 8 Hz, H-13β), 2.12 (1H, dddd, J3β,3R ) 13.2 Hz, J3β,2R ) 2.9 Hz, J3β,2β ) 3.4 Hz, J3β,1β ) 1.3 Hz, H-3β), 2.00 (2H, m*, H-6R and H-14R), 1.91 (1H, dtt, J2β,2R ) 13.4 Hz, J2β,1R ) J2β,3R ) 13.2 Hz, J2β,1β ) J2β,3β ) 3.4 Hz, H-2β), 1.84 (1H, m*, H-1β), 1.79 (1H, ddd, J6β,6R ) 13.1 Hz, J6β,5R ) 12.6 Hz, J6β,7R ) 11.7 Hz, H-6β), 1.67 (3H, d, J17,15 ) 1.5 Hz, Me-17), 1.61 (1H, br d, J14β,14R ) 10.2 Hz, J14β,13β < 0.5 Hz, H-14β), 1.56 (2H, m*, H-11R and H-11β), 1.50 (2H, m*, H-12R and H-12β), 1.38 (1H, ddddd, J2R,2β ) 13.4 Hz, J2R,1R ) 3.9 Hz, J2R,1β ) 3.1 Hz, J2R,3R ) 4.2 Hz, J2R,3β ) 2.9 Hz, H-2R), 1.19 (3H, s, Me-18), 1.11 (1H, dd, J5R,6R ) 2.1 Hz, J5R,6β ) 12.6 Hz, H-5R), 1.00 (1H, td, J3R,3β ) J3R,2β ) 13.2 Hz, J3R,2R ) 4.2 Hz, H-3R), 0.98 (3H, s, Me-20), 0.93 (1H, br d, J9R,11R ) 7.8 Hz, J9R,11β < 0.5 Hz, H-9R), 0.79 (1H, td, J1R,1β ) J1R,2β ) 13.2 Hz, J1R,2R ) 3.9 Hz, H-1R);43 13C NMR (acetone-d6, 100 MHz), see Table 1; EIMS m/z 318 [M]+ (65), 303 (15), 300 (14), 290 (3), 285 (5), 272 (22), 229 (11), 223 (11), 207 (10), 164 (21), 157 (11), 147 (26), 131 (21), 123 (61), 121 (54), 118 (43), 109 (51), 107 (52), 105 (40), 94 (100); anal. C 75.30%, H 9.64%, calcd for C20H30O3, C 75.43%, H 9.50%. ent-12β-Acetoxy-17-oxokaur-15-en-19-oic acid (7):28 amorphous white solid, mp 90-100 °C; [R]D20 -37.2° (c 0.326, CHCl3); IR (KBr) νmax 3426, 2930, 2851, 2725, 1733, 1693, 1678, 1607, 1445, 1369, 1238, 1209, 1027, 986, 974, 854, 754 cm-1; 1H NMR (CDCl , 300 MHz) δ 9.70 (1H, s, H-17), 6.61 (1H, s, 3 H-15), 4.91 (1H, br dd, J12R,11R ) 6.2 Hz, J12R,11β < 0.5 Hz, J12R,13β ) 3.4 Hz, H-12R), 3.08 (1H, br dd, J13β,12R ) 3.4 Hz, J13β,14R ) 4.2 Hz, H-13β), 2.50 (1H, br d, J14β,14R ) 11.3 Hz, J14β,13β = 0 Hz, H-14β), 2.02 (3H, s 12β-OAc), 1.26 (3H, s, Me-18), 1.01 (3H, s, Me-20); EIMS m/z 374 [M]+ (3), 332 (27), 314 (52), 299 (18), 268 (59), 161 (37), 147 (32), 146 (31), 133 (36), 131 (30), 121 (80), 119 (40), 117 (35), 109 (47), 107 (45), 43 (100). ent-7β-Hydroxy-15β,16β-epoxykauran-19-oic acid (9):28 colorless fine needles (EtOAc-n-pentane), mp 246-248 °C and 285-290 °C dec; [R]D20 -41.0° (c 0.441, MeOH); IR (KBr) νmax 3417, 2928, 2869, 1702, 1470, 1251, 1229, 1198, 1044, 903, 842, 790 cm-1; 1H NMR (pyridine-d5, 400 MHz) δ 3.98 (1H, dd, J7R,6R ) 4.0 Hz, J7R,6β ) 11.5 Hz, H-7R), 2.91 (1H, s, H-15R), 2.52 (1H, ddd, J6R,6β ) 13.4 Hz, J6R,5R ) 2.2 Hz, J6R,7R ) 4.0 Hz, H-6R), 2.47 (1H, dddd, J3β,3R ) 13.2 Hz, J3β,2R ) 4.1 Hz, J3β,2β ) 3.7 Hz, J3β,1β ) 1.1 Hz, H-3β), 2.39 (1H, dt, J6β,6R ) 13.4 Hz, J6β,5R ) J6β,7R ) 11.5 Hz, H-6β), 2.26 (1H qt, J2β,2R ) J2β,1R ) J2β,3R ) 13.2 Hz, J2β,1β ) J2β,3β ) 3.7 Hz, H-2β), 2.15 (1H, dd, J14R,14β ) 11.0 Hz, J14R,13β ) 5.4 Hz, H-14R), 2.07 (1H, ddd, J13β,12R ) 4.9 Hz, J13β,12β ) 2.7 Hz, J13β,14R ) 5.4 Hz, J13β,14β = 0 Hz, H-13β), 1.86 (dddd, J1β,1R ) 13.2 Hz, J1β,2R ) 3.2 Hz, J1β,2β ) 3.7 Hz, J1β,3β ) 1.1 Hz, H-1β), 1.52 (1H, m*, H-2R), 1.50 (4H, m*, H-11R, H-11β, H-12R, and H-12β), 1.41 (3H, s, Me-17), 1.34 (3H, s, Me-18), 1.30 (1H, br d, J14β,14R ) 11.0 Hz, J14β,13β
Labdane and Kaurane Diterpenoids from Plectranthus
= 0 Hz, H-14β), 1.20 (1H, dd, J5R,6R ) 2.2 Hz, J5R,6β ) 11.5 Hz, H-5R), 1.15 (3H, s, Me-20), 1.14 (1H, br d, J9R,11R ) 7.0 Hz, J9R,11β < 0.5 Hz, H-9R), 1.07 (1H, td, J3R,3β ) J3R,2β ) 13.2 Hz, J3R,2R ) 4.3 Hz, H-3R), 0.86 (1H, td, J1R,1β ) J1R,2β ) 13.2 Hz, J1R,2R ) 3.9 Hz, H-1R);43 13C NMR (pyridine-d5, 100 MHz), see Table 1; EIMS m/z 334 [M]+ (100), 319 (10), 316 (65), 301 (25), 289 (44), 271 (38), 255 (32), 159 (26), 151 (35), 149 (29), 147 (34), 145 (33), 137 (52), 136 (57), 135 (56), 133 (44), 131 (34), 125 (31), 123 (71), 121 (44), 119 (44), 109 (58), 107 (63), 43 (82); anal. C 71.96%, H 8.89%, calcd for C20H30O4, C 71.82%, H 9.04%. ent-Labda-8(17),12Z,14-triene-2r,3β-diol (10):28 amorphous white solid, mp 74-80 °C; [R]D20 -22.4° (c 0.49, CHCl3); IR, 1H and 13C NMR, and mass spectra identical to those reported2 for the compound isolated from Croton joufra [mp 72-74 °C; [R]D25 -18.24° (c 0.34, CHCl3)] and for the ent-2Rdeacetyl derivative of 15 [mp 76-80 °C; [R]D20 -23.7° (c 0.313, CHCl3)].1 Methyl ent-12β-hydroxykaur-16-en-19-oate (12):28 obtained by methylation of 11; colorless thick oil; [R]D18 -57.1° (c 0.621, CHCl3); IR, 1H NMR, and mass spectra identical to those reported previously.3-6 Lit.: thick oil,3,5 no [R]D value has previously been reported.3-6 For ent-12β-hydroxykaur-16en-19-oic acid: [R]D24 -44.7° (c 1.0, CHCl3).5 Methyl ent-12β-acetoxy-7β-hydroxykaur-15-en-19-oate (13):28 colorless prisms (EtOAc-n-pentane), mp 113-115 °C; [R]D18 -14.8° (c 0.446, CHCl3); IR (KBr) νmax 3542, 3021, 2943, 1723, 1708, 1635, 1467, 1437, 1369, 1241, 1155, 1034, 1016, 994, 965, 817 cm-1; 1H NMR (CDCl3, 400 MHz) δ 5.19 (1H, qd, J15,17 ) 1.6 Hz, J15,14β ) 0.8 Hz, H-15), 4.94 (1H, ddd, J12R,11R ) 6.9 Hz, J12R,11β ) 1.0 Hz, J12R,13β ) 3.2 Hz, H-12R), 3.63 (3H, s, 19-COOMe), 3.60 (1H, dd, J7R,6R ) 4.0 Hz, J7R,6β ) 12.4 Hz, H-7R), 2.48 (1H, br dd, J13β,12R ) 3.2 Hz, J13β,14R ) 4.0 Hz, J13β,14β = 0 Hz, H-13β), 2.17 (1H, dddd, J3β,3R ) 13.5 Hz, J3β,2R ) 4.2 Hz, J3β,2β ) 3.8 Hz, J3β,1β ) 1.6 Hz, H-3β), 2.07 (1H, ddd, J6R,6β ) 13.1 Hz, J6R,5R ) 1.8 Hz, J6R,7R ) 4.0 Hz, H-6R), 2.02 (3H, s, 12β-OAc), 1.97 (1H, ddd, J11R,11β ) 17.0 Hz, J11R,9R ) 9.9 Hz, J11R,12R ) 6.9 Hz, H-11R), 1.95 (1H, br dd, J14β,14R ) 11.7 Hz, J14β,13β = 0 Hz, J14β,15 ) 0.8 Hz, H-14β), 1.80 (1H, dddt, J2β,2R ) J2β,3R ) 13.4 Hz, J2β,1R ) 13.2 Hz, J2β,1β ) J2β,3β ) 3.8 Hz, H-2β), 1.77 (3H, d, J17,15 ) 1.6 Hz, Me-17), 1.75 (1H, ddd, J6β,6R ) 13.1 Hz, J6β,5R ) 12.5 Hz, J6β,7R ) 12.4 Hz, H-6β), 1.70 (1H, dd, J14R,14β ) 11.7 Hz, J14R,13β ) 4.0 Hz, H-14R), 1.68 (1H, dddd, J1β,1R ) 13.2 Hz, J1β,2R ) 3.5 Hz, J1β,2β ) 3.8 Hz, J1β,3β ) 1.6 Hz, H-1β), 1.56 (1H, br s, 7β-OH), 1.53 (1H, br dd, J11β,11R ) 17.0 Hz, J11β,9R < 0.5 Hz, J11β,12R ) 1.0 Hz, H-11β), 1.40 (1H, ddddd, J2R,2β ) 13.4 Hz, J2R,1R ) 4.1 Hz, J2R,1β ) 3.5 Hz, J2R,3R ) 4.3 Hz, J2R,3β ) 4.2 Hz, H-2R), 1.17 (3H, s, Me-18), 1.10 (1H, br d, J9R,11R ) 9.9 Hz, J9R,11β < 0.5 Hz, H-9R), 1.07 (1H, dd, J5R,6R ) 1.8 Hz, J5R,6β ) 12.5 Hz, H-5R), 0.96 (1H, td, J3R,3β ) J3R,2β ) 13.5 Hz, J3R,2R ) 4.3 Hz, H-3R), 0.87 (3H, s, Me-20), 0.72 (1H, td, J1R,1β ) J1R,2β ) 13.2 Hz, J1R,2R ) 4.1 Hz, H-1R); 13C NMR (CDCl , 100 MHz), see Table 1; EIMS m/z 390 [M]+ 3 (10), 375 (1), 372 (1), 348 (2), 330 (18), 315 (12), 312 (32), 287 (14), 271 (13), 253 (14), 237 (24), 162 (45), 145 (56), 144 (49), 131 (33), 123 (89), 121 (42), 119 (48), 109 (80), 107 (66), 43 (100); anal. C 70.81%, H 8.69%, calcd for C23H34O5, C 70.74%, H 8.78%. Methyl ent-12β-acetoxy-15β,16β-epoxykauran-19-oate (14):28 colorless prisms (EtOAc-n-hexane), mp 195-196 °C; [R]D20 -17.4° (c 1.196, CHCl3); IR (KBr) νmax 3010, 2998, 2951, 1720, 1434, 1363, 1239, 1157, 1029, 990, 851 cm-1; 1H NMR (CDCl3, 400 MHz) δ 4.97 (1H, ddd, J12R,11R ) 4.8 Hz, J12R,11β ) 1.9 Hz, J12R,13β ) 4.1 Hz, H-12R), 3.63 (3H, s, 19-COOMe), 2.63 (1H, s, H-15R), 2.23 (1H, t, J13β,12R ) J13β,14R ) 4.1 Hz, J13β14β = 0 Hz, H-13β), 2.16 (1H, ddd, J3β,3R ) 13.3 Hz, J3β,2R ) 4.2 Hz, J3β,2β ) 3.6 Hz, H-3β), 2.01 (3H, s, 12β-OAc), 1.82 (1H, m*, H-6R), 1.78 (4H, m*, H-2β, H-7β, H-11R, and H-14β), 1.71 (1H, ddd, J1β,1R ) 13.4 Hz, J1β,2R ) 4.2 Hz, J1β,2β ) 3.6 Hz, H-1β), 1.63 (1H, m*, H-11β), 1.61 (1H, m*, H-6β), 1.47 (1H, ddd, J7R,7β ) 13.3 Hz, J7R,6R ) 2.4 Hz, J7R,6β ) 12.0 Hz, H-7R), 1.43 (3H, s, Me-17), 1.39 (1H, dm, J2R,2β ) 13.1 Hz, J2R,1R = J2R,1β = J2R,3R = J2R,3β = 4 Hz, H-2R), 1.23 (1H, br d, J9R,11R ) 9.6 Hz, J9R,11β < 0.5 Hz, H-9R), 1.16 (3H, s, Me-18), 1.01 (1H, dd, J5R,6R ) 1.4 Hz, J5R,6β ) 11.5 Hz, H-5R), 0.98 (1H, td, J3R,3β ) J3R,2β ) 13.3
Journal of Natural Products, 2004, Vol. 67, No. 4 619
Hz, J3R,2R ) 4.2 Hz, H-3R), 0.93 (1H, dd, J14R,14β ) 12.0 Hz, J14R,13β ) 4.1 Hz, H-14R), 0.83 (3H, s, Me-20), 0.79 (1H, td, J1R,1β ) J1R,2β ) 13.4 Hz, J1R,2R ) 4.1 Hz, H-1R);43 13C NMR (CDCl3, 100 MHz), see Table 1; EIMS m/z 390 [M]+ (5), 375 (1), 347 (100), 331 (27), 330 (30), 315 (17), 287 (42), 271 (41), 255 (20), 227 (29), 149 (25), 147 (29), 145 (25), 135 (36), 131 (26), 121 (57), 119 (26), 109 (31), 107 (30), 43 (87); anal. C 70.81%, H 8.92%, calcd for C23H34O5, C 70.74%, H 8.78%. Preparation of Compound 10 from Compound 2. A stirred solution of 2 (8 mg, 0.023 mmol) in EtOH (2 mL) was treated with an ethanolic solution of KOH (8%, w/v, 1.5 mL, 2.14 mmol) at room temperature for 18 h. Then, water (10 mL) was added to the reaction and the mixture was extracted with CH2Cl2 (10 × 4 mL). The extracts were dried (Na2SO4) and filtered, and the solvents removed in vacuo, yielding a residue (4 mg, 0.013 mmol, 56.5%) of pure 10:1,2 amorphous white solid, mp 70-78 °C; [R]D20 -21.8° (c 0.201, CHCl3); 1H NMR (CDCl3, 400 MHz) and mass spectra identical to those obtained for 3 (see above). Benzoylation of Compound 101,2 to Give ent-Labda8(17),12Z,14-triene-2r,3β-dibenzoate (16).28 To a solution of 10 (40 mg, 0.131 mmol) in anhydrous pyridine (4 mL) was added an excess of benzoyl chloride (50 mg, 0.355 mmol), and the reaction mixture was left at room temperature for 5 h. Water (20 mL) was added, and the reaction mixture was stirred for 30 min and then extracted with CH2Cl2 (4 × 20 mL). The extract was washed with a saturated aqueous solution of Na2CO3 (4 × 10 mL), then with water (2 × 10 mL), and finally dried (Na2SO4) and filtered, and the solvents were removed in vacuo. The residue (60 mg) was subjected to column chromatography [Si gel 230-400 mesh, 10 g, petroleum etherEtOAc (49:1) as eluent], yielding pure 16 (43 mg, 0.084 mmol, 63.8%): amorphous white powder, mp 75-85 °C; [R]D19 +28.2° (c 0.305, CHCl3); CD ∆248 -6.6, ∆243 0, ∆235 +12.5, ∆229 0, ∆224 -10.3 (C 10-3 M, dioxane); IR (KBr) νmax 3087, 2973, 2944, 2857, 1722, 1644, 1602, 1584, 1450, 1314, 1280, 1111, 1069, 1026, 993, 953, 895, 709 cm-1; 1H NMR (CDCl3, 400 MHz) δ 6.73 (1H, ddd, J14,15A ) 10.8 Hz, J14,15B ) 17.3 Hz, J14,12 ) 0.7 Hz, H-14), 5.47 (1H, ddd, J2β,1R ) 11.5 Hz, J2β,1β ) 4.5 Hz, J2β,3R ) 10.4 Hz, H-2β), 5.25 (1H, d, J3R,2β ) 10.4 Hz, H-3R), 5.24 (1H, br t, J12,11A ) J12,11B ) 6.4 Hz, H-12), 5.15 (1H, ddd, J15B,15A ) 1.6 Hz, J15B,14 ) 17.3 Hz, J15B,12 ) 0.6 Hz, HB-15, pro-Z hydrogen), 5.07 (1H, dt, J15A,14 ) 10.8 Hz, J15A,15B ) J15A,12 ) 1.6 Hz, HA-15, pro-E hydrogen), 4.91 (1H, q, J17A,17B ) J17B,7R ) J17B,9R ) 1.5 Hz, HB-17, pro-E hydrogen), 4.54 (1H, q, J17A,17B ) J17A,7R ) J17A,9R ) 1.5 Hz, HA-17, pro-Z hydrogen), 2.45 (1H, ddd, J7β,7R ) 13.0 Hz, J7β,6R ) 2.4 Hz, J7β,6β ) 4.2 Hz, H-7β), 2.39 (1H, dd, J1β,1R ) 12.4 Hz, J1β,2β ) 4.5 Hz, H-1β), 2.35 (1H, ddd, J11B,11A ) 17.5 Hz, J11B,9R ) 11.2 Hz, J11B,12 ) 6.4 Hz, HB11), 2.25 (1H, ddd, J11A,11B ) 17.5 Hz, J11A,9R ) 3.2 Hz, J11A,12 ) 6.4 Hz, HA-11), 2.06 (1H, br ddd, J7R,7β ) 13.0 Hz, J7R,6R ) 4.0 Hz, J7R,6β ) 12.0 Hz, H-7R), 1.85 (1H, br dd, J9R,11A ) 3.2 Hz, J9R,11B ) 11.2 Hz, H-9R), 1.79 (1H, dddd, J6R,6β ) 12.9 Hz, J6R,5R ) 2.8 Hz, J6R,7R ) 4.0 Hz, J6R,7β ) 2.4 Hz, H-6R), 1.75 (3H, d, J16,12 ) 1.1 Hz, Me-16), 1.52 (1H, dd, J1R,1β ) 12.4 Hz, J1R,2β ) 11.5 Hz, H-1R), 1.48 (2H, m, H-5R and H-6β, these assignments were in agreement with the HSQC spectrum), 1.09 (3H, s, Me-19), 1.01 (3H, s, Me-18), 0.99 (3H, s, Me-20), 2R-OBz: 7.89 (2H, dd, J ) 8.4, 1.4 Hz, H-2′ and H-6′), 7.32 (2H, dd, J ) 8.4, 7.4 Hz, H-3′ and H-5′), 7.44 (1H, tt, J ) 7.4, 1.4 Hz, H-4′), 3β-OBz: 7.96 (2H, dd, J ) 8.4, 1.4 Hz, H-2′′ and H-6′′), 7.34 (2H, dd, J ) 8.4, 7.4 Hz, H-3′′ and H-5′′), 7.46 (1H, tt, J ) 7.4, 1.4 Hz, H-4′′); 13C NMR (CDCl3, 100 MHz) δ 146.6 (C, C-8), 133.7 (CH, C-14), 132.1 (C, C-13), 130.5 (CH, C-12), 113.6 (CH2, C-15), 109.3 (CH2, C-17), 80.6 (CH, C-3), 71.1 (CH, C-2), 56.8 (CH, C-9), 54.4 (CH, C-5), 42.5 (CH2, C-1), 40.2 (C, C-10), 39.8 (C, C-4), 37.5 (CH2, C-7), 28.7 (CH3, C-18), 23.5 (CH2, C-6), 22.3 (CH2, C-11), 19.7 (CH3, C-16), 17.7 (CH3, C-19), 15.4 (CH3, C-20), OBz: 166.2 and 166.4 (C, OCOC6H5), 132.9 and 132.8 (CH, C-4′ and C-4′′), 130.1 and 129.9 (C, C-1′ and C-1′′), 129.5 (CH, C-2′, C-6′, C-2′′, and C-6′′), 128.3 and 128.2 (CH, C-3′, C-5′, C-3′′, and C-5′′); EIMS m/z 512 [M]+ (0.1), 390 (1.4), 375 (0.3), 268 (4), 253 (5), 187 (6), 133 (6), 119 (5), 105 (100); anal. C 79.40%, H 7.71%, calcd for C34H40O4, C 79.65%, H 7.86%.
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Methylation of Compound 3 to Give Methyl ent-12βAcetoxy-15β-hydroxykaur-16-en-19-oate (17).28 A solution of 3 (25 mg, 0.066 mmol) in Et2O (50 mL) was treated with an excess of an ethereal solution of CH2N2 at room temperature for 3 h. After evaporation of the solvent a residue (25 mg) remained. Crystallization from EtOAc-n-pentane yielded 17 (22 mg, 0.056 mmol, 84.8%): colorless plates, mp 128-131 °C; [R]D20 -43.0° (c 0.293, CHCl3); IR (KBr) νmax 3444, 3071, 2949, 1726, 1632, 1464, 1376, 1240, 1151, 1017, 908 cm-1; 1H NMR (CDCl3, 400 MHz) δ 5.30 (1H, br d, J17B,17A ) 1.1 Hz, HB-17, cis hydrogen with respect to C-15), 5.23 (1H, br d, J17A,17B ) 1.1 Hz, HA-17, trans hrydrogen with respect to C-15), 4.72 (1H, ddd, J12R,11R ) 4.2 Hz, J12R,11β ) 1.2 Hz, J12R,13β ) 4.4 Hz, H-12R), 3.83 (1H, s, H-15R), 3.65 (3H, s, 19-COOMe), 2.84 (1H, br t, J13β,12R = J13β,14R = 4.5 Hz, J13β,14β < 0.5 Hz, H-13β), 2.16 (1H, dddd, J3β,3R ) 13.3 Hz, J3β,2R ) 4.3 Hz, J3β,2β ) 3.8 Hz, J3β,1β ) 1.8 Hz, H-3β), 2.15 (1H, br d, J14β,14R ) 12.3 Hz, J14β,13β < 0.5 Hz, H-14β), 2.02 (3H, s, 12β-OAc), 1.91 (1H, dddd, J6R,6β ) 13.6 Hz, J6R,5R ) 2.1 Hz, J6R,7R ) 4.0 Hz, J6R,7β ) 3.4 Hz, H-6R), 1.78 (2H, m*, H-2β and H-7β), 1.70 (3H, m*, H-1β, H-6β, and H-11R), 1.61 (1H, br dd, J11β,11R ) 16.4 Hz, J11β,9R < 0.5 Hz, J11β,12R ) 1.2 Hz, H-11β), 1.40 (1H, td, J7R,7β ) J7R,6β ) 13.6 Hz, J7R,6R ) 4.0 Hz, H-7R), 1.39 (1H, m*, H-2R), 1.30 (1H, dd, J14R,14β ) 12.3 Hz, J14R,13β ) 4.6 Hz, H-14R), 1.18 (3H, s, Me18), 1.14 (1H, br d, J9R,11R ) 9.6 Hz, J9R,11β < 0.5 Hz, H-9R), 1.04 (1H, dd, J5R,6R ) 2.1 Hz, J5R,6β ) 12.1 Hz, H-5R), 0.98 (1H, td, J3R,3β ) J3R,2β ) 13.3 Hz, J3R,2R ) 4.2 Hz, H-3R), 0.87 (3H, s, Me-20), 0.75 (1H, td, J1R,1β ) J1R,2β ) 13.2 Hz, J1R,2R ) 4.2 Hz, H-1R);43 13C NMR (CDCl3, 100 MHz), see Table 1; EIMS m/z 390 [M]+ (1), 372 (1), 330 (100), 312 (17), 271 (42), 270 (49), 255 (29), 253 (27), 237 (26), 173 (23), 161 (31), 148 (37), 147 (31), 145 (35), 133 (34), 131 (34), 123 (43), 121 (70); anal. C 70.66%, H 8.90%, calcd for C23H34O5, C 70.74%, H 8.78%. Methylation of Compound 7 to Give Methyl ent-12βAcetoxy-17-oxokaur-15-en-19-oate (19).28 Treatment of 7 (20 mg, 0.053 mmol) with an excess of CH2N2, as described above for obtaining 17, yielded the methyl ester 19 (16 mg, 0.041 mmol, after crystallization from EtOAc-n-pentane, 77.4%): colorless plates, mp 146-149 °C; [R]D20 -39.6° (c 0.723, CHCl3); UV (MeOH) λmax (log ) 249 (3.84) nm; IR (KBr) νmax 2990, 2955, 2730, 2708, 1738, 1713, 1684, 1610, 1444, 1370, 1243, 1208, 1026, 985, 973 cm-1; 1H NMR (CDCl3, 400 MHz) δ 9.69 (1H, d, J17,15 ) 0.8 Hz, H-17), 6.60 (1H, d, J15,17 ) 0.8 Hz, H-15), 4.90 (1H, ddd, J12R,11R ) 6.0 Hz, J12R,11β ) 1.2 Hz, J12R,13β ) 4.2 Hz, H-12R), 3.64 (3H, s, 19-COOMe), 3.07 (1H, t, J13β,12R ) J13β,14R ) 4.2 Hz, J13β,14β ) 0 Hz, H-13β), 2.47 (1H, d, J14β,14R ) 11.4 Hz, J14β,13β ) 0 Hz, H-14β), 2.16 (1H, dddd, J3β,3R ) 13.4 Hz, J3β,2R ) 4.2 Hz, J3β,2β ) 3.8 Hz, J3β,1β ) 1.6 Hz, H-3β), 2.02 (3H, s, 12β-OAc), 1.86 (1H, m*, H-6R), 1.82 (1H, m*, H-11R), 1.80 (1H, m*, H-2β), 1.74 (1H, m*, H-7β), 1.70 (1H, m*, H-6β), 1.69 (1H, m*, H-1β), 1.69 (1H, td, J7R,7β ) J7R,6β ) 13.0 Hz, J7R,6R ) 4.0 Hz, H-7R), 1.61 (1H, br dd, J11β,11R ) 17.6 Hz, J11β,9R < 0.5 Hz, J11β,12R ) 1.2 Hz, H-11β), 1.40 (1H, m*, H-2R), 1.38 (1H, m*, H-14R), 1.23 (1H, br d, J9R,11R ) 9.6 Hz, J9R,11β < 0.5 Hz, H-9R), 1.17 (3H, s, Me-18), 1.07 (1H, dd, J5R,6R ) 2.8 Hz, J5R,6β ) 11.6 Hz, H-5R), 0.98 (1H, td, J3R,3β ) J3R,2β ) 13.4 Hz, J3R,2R ) 4.2 Hz, H-3R), 0.87 (3H, s, Me-20), 0.76 (1H, td, J1R,1β ) J1R,2β ) 13.2 Hz, J1R,2R ) 4.1 Hz, H-1R);43 13C NMR (CDCl3, 100 MHz), see Table 1; EIMS m/z 388 [M]+ (4), 346 (19), 328 (57), 316 (27), 269 (43), 268 (58), 257 (22), 253 (21), 173 (23), 161 (46), 160 (30), 147 (30), 133 (35), 123 (57), 121 (97), 119 (44), 117 (36), 109 (62), 107 (63), 43 (100); anal. C 70.93%, H 8.21%, calcd for C23H32O5, C 71.11%, H 8.30%. Biological Assays. Antimicrobial activities of 1, 3-5, 7, 9, 10, 12-14, 17, and 19 were tested against Staphylococcus aureus ATCC 25923, Escherischia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Candida albicans CIP 3153A. The minimum inhibitory concentration (MIC) values were determined by the serial broth microdilution method according to NCCLS.44 The compounds were dissolved in DMSO and graded concentration of broth medium (MuellerHinton for bacteria, YMA for the yeast) ranging from 250 to 7.8 µg/mL. Solvent blank was included. Kanamycin was used as positive control (MIC values < 7.8 mg/mL for S. aureus and E. coli strains).
Gaspar-Marques et al.
Acknowledgment. The authors are indebted to Prof. R. Riguera, University of Santiago de Compostela, Spain, for recording the CD spectrum of 14 and helpful discussions. Thanks to Dr. E. S. Martins, “Centro de Botaˆnica do Instituto de Investigac¸ a˜o Cientı´fica Tropical”, Lisbon, Portugal, for the identification of the plant material. The authors are also very grateful to Prof. A. Duarte, Microbiology Laboratory, Faculty of Phamacy, Lisbon University, Portugal, for performing the biological assay. This work was supported by funds from the Spanish “Comisio´n Interministerial de Ciencia y Tecnologı´a” (CICYT, grant no. AGL 2001-1652) and from Portuguese FCT (I&D no. 8/94), POCTI (QCA III), and Feder Porjects. One of us (C.G.-M.) thanks the Praxis Program for a fellowship (Praxis XXI/BD/18046/98). References and Notes (1) Gaspar-Marques, C.; Simo˜es, M. F.; Duarte, A.; Rodrı´guez, B. J. Nat. Prod. 2003, 66, 491-496. (2) Sutthivaiyakit, S.; Nareeboon, P.; Ruangrangsi, N.; Ruchirawat, S.; Pisutjaroenpong, S.; Mahidol, C. Phytochemistry 2001, 56, 811-814. (3) Beale, M. H.; Bearder, J. R.; MacMillan, J.; Matsuo, A.; Phinney, B. O. Phytochemistry 1983, 22, 875-881. (4) Lewis, N. J.; MacMillan, J. J. Chem. Soc., Perkin Trans. 1 1980, 1270-1278. (5) Bohlmann, F.; Knoll, K.-H.; Robinson, H.; King, R. M. Phytochemistry 1980, 19, 107-110. (6) Bohlmann, F.; Jakupovic, J.; King, R. M.; Robinson, H. Phytochemistry 1980, 19, 863-868. (7) Biftu, T.; Stevenson, R. J. Chem. Soc., Perkin Trans. 1 1978, 360363. (8) Star, A. E.; Mabry, T. J. Phytochemistry 1971, 10, 2817-2818. (9) Sakakibara, M.; DiFeo, D.; Nakatani, N.; Timmermann, B.; Mabry, T. J. Phytochemistry 1976, 15, 727-731. (10) Wollenweber, E.; Wassum, M. Tetrahedron Lett. 1972, 9, 797-800. (11) Rodrı´guez, B.; Martı´n-Panizo, F. An. Quı´m. 1979, 75, 431-432. (12) Rao, M. M.; Kingston, D. G. I.; Spittler, T. D. Phytochemistry 1970, 9, 227-228. (13) Kupchan, S. M.; Sigel, C. W.; Hemingway, R. J.; Knox, J. R.; Udayamurthy, M. S. Tetrahedron 1969, 25, 1603-1615. (14) Timmermann, B. N.; Mues, R.; Mabry, T. J.; Powell, A. M. Phytochemistry 1979, 18, 1855-1858. (15) Seebacher, W.; Simic, N.; Weis, R.; Saf, R.; Kunert, O. Magn. Reson. Chem. 2003, 41, 636-638. (16) Krebs, H. C.; Rakotoarimanga, J. V.; Habermehl, G. G. Magn. Reson. Chem. 1990, 28, 124-128. (17) Juell, S. M.-K.; Hansen, R.; Jork, H. Arch. Pharm. (Weinheim, Ger.) 1976, 309, 458-466. (18) Several numbering systems are in use for aromadendranes; the one chosen for this paper is that most commonly used (Connolly, J. D.; Hill, R. A. Dictionary of Terpenoids; Chapman & Hall: London, 1991; Vol. 1, p XXV), and it is indicated on formula 1. (19) Faure, R.; Ramanoelina, A. R. P.; Rakotonirainy, O.; Bianchini, J.P.; Gaydou, E. M. Magn. Reson. Chem. 1991, 29, 969-971. (20) Ciminiello, P.; Fattorusso, E.; Magno, S.; Mayol, L. Can. J. Chem. 1987, 65, 518-522. (21) Rashid, M. A.; Gray, A. I.; Waterman, P. G.; Armstrong, J. A. J. Nat. Prod. 1995, 58, 618-621. (22) Wessels, M.; Ko¨nig, G. M.; Wright, A. D. J. Nat. Prod. 2001, 64, 370372. (23) Goldsby, G.; Burke, B. A. Phytochemistry 1987, 26, 1059-1063. (24) Gijsen, H. J. M.; Wijnberg, J. B. P. A.; de Groot, Ae. In Progress in the Chemistry of Organic Natural Products; Herz, W., Kirby, G. W., Moore, R. E., Steglich, W., Tamm, Ch., Eds.; Springer-Verlag: Vienna, 1995; Vol. 64, pp 149-193. (25) Abdel-Mogib, M.; Albar, H. A.; Batterjee, S. M. Molecules 2002, 7, 271-301. (26) Fournier, G.; Paris, M.; Dumitresco, S. M.; Pages, N.; Boudene, C. Planta Med. 1986, 52, 486-488. (27) A GC analysis26 of the essential oil of P. fruticosus on a fused silica capillary column allowed the identification of 10(14)-aromadendrene, but without defining its absolute configuration. (28) Since compounds 2-17 and 19 belong to the enantio series, the entR- or ent-β-configuration for a substituent indicates that it is placed, respectively, above or below the plane of the formulas depicted for these substances. Moreover, the R- or β-configurations indicated for the assignment of the 1H NMR spectra of these compounds also refer to this nomenclature throughout the text. (29) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy. Exciton Coupling in Organic Stereochemistry; University Science Books: Mill Valley, CA, 1983; pp 15-17, 55-103. (30) Compound 16 showed another Cotton effect (∆248 -6.6) which could be associated with interactions between the benzoates and the 12Z,14-diene chromophore (see ref 29, pp 255-259). (31) Hutchison, M.; Lewer, P.; MacMillan, J. J. Chem. Soc., Perkin Trans. 1 1984, 2363-2366. (32) Liu, G.; Mu¨ller, R.; Ru¨edi, P. Helv. Chim. Acta 2003, 86, 420-438.
Labdane and Kaurane Diterpenoids from Plectranthus (33) Connolly, J. D.; Hill, R. A. Dictionary of Terpenoids; Chapman & Hall: London, 1991; Vol. 2, pp 906-946, 948-956. (34) Katti, S. B.; Ru¨edi, P.; Eugster, C. H. Helv. Chim. Acta 1982, 65, 2189-2197. (35) Liu, G.; Ru¨edi, P. Phytochemistry 1996, 41, 1563-1568. (36) Fujita, E.; Ochiai, M.; Ichida, I.; Chatterjee, A.; Desmukh, S. K. Phytochemistry 1975, 14, 2249-2251. (37) Only eight phyllocladane diterpenoids have been isolated from Plectranthus species,25,32-34 whereas 21 ent-kaurane derivatives, including those described in this work (3-9 and 11), have been found1,25 in plants belonging to this genus. (38) Duc, D. K. M.; Fe´tizon, M.; Lazare, S.; Grant, P. K.; Nicholls, M. J.; Liau, H. T. L.; Francis, M. J.; Poisson, J.; Bernassau, J.-M.; Roque, N. F.; Wovkulich, P. M.; Wenkert, E. Tetrahedron 1981, 37, 23712374.
Journal of Natural Products, 2004, Vol. 67, No. 4 621 (39) Bohlmann, F.; Suding, H.; Cuatrecasas, J.; Robinson, H.; King, R. M. Phytochemistry 1980, 19, 2399-2403. (40) Herz, W.; Kulanthaivel, P.; Watanabe, K. Phytochemistry 1983, 22, 2021-2025. (41) Harrigan, G. G.; Bolzani, V. S.; Gunatilaka, A. A. L.; Kingston, D. G. I. Phytochemistry 1994, 36, 109-113. (42) Anam, E. M. Indian. J. Chem. 1998, 37B, 187-189. (43) Signals marked with an asterisk appeared as overlapped or partially overlapped multiplets, and their assignments were in agreement with the HSQC spectrum. (44) Anonymous. In National Committee for Clinical Laboratory Standards, 5th ed.; NCCLS: Wayne, PA, 2000; approved standard M7-A5.
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